The transforming growth factor beta (TGF-β) family forms a group of three isoforms, TGF-β1, TGF-β2, and TGF-β3, with their structure formed by interrelated dimeric polypeptide chains. Pleiotropic and redundant functions of the TGF-β family relate to control of numerous aspects and effects of cell functions in all tissues of the human body, including aspects of proliferation, differentiation, and migration (Poniatowski L A, et al, 2015, Mediators Inflamm, 2015; 137823). Although the isoforms are similar in sequence (TGF-ß3 active domain shares 86% similarity with TGF-ß1 and 91% with TGF-ß2), protein crystal structure and NMR studies have shown that TGF-ß3 active domain structure is different from TGF-ß1. Comparison of the TGF-ß3 with the structure of TGF-ß2 (Schlunegger M P, Grafter M G, 1992, Nature 358:430-434; Daopin S, Piez K A, Ogawa Y, Davies D R, 1992, Science 257:369-373) reveals a virtually identical central core. Differences exist in the conformations of the N-terminal alpha-helix and in the beta-sheet loops (Mittl PR1, Priestle J P, Cox D A, McMaster G, Cerletti N, Grafter M G, 1996, Protein Science July 5 (7): 1261-1271).
In most cells, three types of cell surface proteins mediate TGF-β signaling: TGF-β receptor I (TβRI), II (TβRII) and III (TβRIII) (Cheifetz S, Like B, Massagué J, J Biol Chem. 1986 Jul. 25; 261(21):9972-8). Bioactive forms of TGF-βs are dimers held together by hydrophobic interactions and, in most cases, by an intersubunit disulfide bond as well. The dimeric structure of these ligands suggests that they function by bringing together pairs of type I and II receptors, forming heterotetrameric receptor complexes (Sun P D, Davies D R, Annu Rev Biophys Biomol Struct. 1995; 24:269-91). Binding of TGF-β to extracellular domains of both receptors also induces proper conformation of the intracellular kinase domains. These receptors are subject to reversible post-translational modifications (phosphorylation, ubiquitylation and sumoylation) that regulate stability and availability of receptors as well as SMAD and non-SMAD pathway activation.
Receptor phosphorylation activates the TGF-β signaling pathway—the ligand binds to TβRII first, followed by subsequent phosphorylation of a Gly-Ser regulatory region (GS-domain) within TβRI. This leads to incorporation of TβRI and formation of a large ligand-receptor complex that consists of dimeric TGF-β ligand and two pairs of TβRI and TβRII (Shi Y, Massagué J, Cell. 2003 Jun. 13; 113(6):685-700). TGF-β1 and TGF-β3 bind to TβRII without participation of type I receptor, whereas TGF-β2 interacts only with combination of both receptors (Derynck R, Feng X H, Biochim Biophys Acta. 1997 Oct. 24; 1333(2):F105-50). It has been observed that different ligand/receptor engagements of the TGF-β family may contribute to qualitative and quantitative differences in signaling events and biological outcomes (Hart P J et al Nat Struct Biol 2002 9(3):203-208). Furthermore, temporal-spatial expression of some of the TGF-β isoforms in embryogenesis is very different, indicating uncompensated, non-overlapping functions throughout development (Akhurst R J et al Development 1990 110(2):445-460).
Overexpression of transforming growth factor β (TGF-β) is frequently associated with tumor metastasis and poor prognosis in animal models of cancer and cancer patients (Donkor M K et al., 2012, OncoImmunology, 1(2):162-171). Members of the TGF-β family are potent regulatory cytokines that affect multiple cell types of the immune system mediating pro-inflammatory or anti-inflammatory responses. The effect of TGF-β on T-cells is highly versatile. In concert with other soluble factors, it controls the maturation, differentiation and activity of various T cell subsets that either prevent or actuate infections, graft-versus-host reactions, immune diseases, and cancer formation (Schon H T et al., 2014, Hepatobiliary Surg Nutr, 2014, Dec. 3(6):386-406).
Studies have demonstrated that blockade of TGF-β, using mouse TGF-β generic antibody 1D11 (which recognizes TGF-β1, TGF-β2 and TGF-β3), synergistically enhances tumor vaccines in animal models via CD8+ T cells (Terabe M et al (2009) Clin Cancer Res 15:6560-6569; Takaku S et al (2010) Int J Cancer 126(7):1666). Also, TGFβ production by tumor cells and by myeloid-derived suppressor cells (MDSC) present at tumor sites along with TGFβ immune suppressive activity at the tumor site implicates blocking TGFβ to enhance antigen uptake, presentation, and activation of antitumor immune response mediated by therapeutic vaccines.
Several publications show differences in melanoma-associated expression of TGF-ß isoforms. Van Belle et al showed that TGF-ß1 is expressed by some melanocytes and almost uniformly by nevi and melanomas while TGF-ß2 and TGF-ß3 were not detected in normal melanocytes but were found in nevi and in all forms of melanomas (early and advanced primary and metastatic melanomas) in a tumor progression related manner. They state that “TGF-ß2 was heterogeneously expressed in advanced primary and metastatic melanomas whereas TGF-ß3 was uniformly and highly expressed in these lesions” (P. Van Belle 1996 American J. of Pathology 148(6): 1887-1894).
Also, TGF-ß3 but not TGF-ß1 immunostaining was reported to correlate in breast carcinomas with poor survival prognosis, and when combined with lymph node involvement, TGF-ß3 was a highly significant prognostic factor for survival (Ghellal A1 2000 Anticancer Res 20: 4413). Moreover, plasma levels of TGF-ß3 and complexes of TGF-ß3 and its receptor CD105 (TGF-ß3-CD105) were significantly elevated in breast cancer patients with positive lymph nodes compared to those without node metastasis, and their levels correlated with lymph node status (Li Cl 1998 Int. J. Cancer 79:455).
Particularly, studies have demonstrated TGF-ß3's involvement in the following: contributing to epithelial mesenchymal transition (EMT); elevated TGF-ß3 levels in breast cancer and prostate metastasis; and elevated levels of TGF-ß3 detected in late stage tumors and aggressive tumors such as breast, prostate, and lung.
Thus, it is apparent that, by targeting specific isoforms of TGF-ß, one could avoid damaging inflammatory consequences of blocking all isoforms of TGF-ß. Moreover, the differential expression patterns of TGF-ß isoforms in different cancer types gives researchers a unique opportunity to target cancer cells more specifically and with greater efficacy. There is an unmet need in the field to generate therapeutic TGF-ß antibodies against its isoforms, including particularly against TGF-β3. In addition, the tools developed for recognizing different TGF-ß isoforms are powerful diagnostic and prognostic sources. The present invention addresses such unmet needs in the field and particularly with regard to TGF-β3.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.